PROJECT SUMMARY Human environmental exposures to heavy metals such as manganese (Mn) or cadmium (Cd) are common and represent adverse risks to health. Mn and Cd generate neurotoxicity by inducing oxidative stress and neuroinflammation. The paraoxonases (PON1, 2 and 3) are potent antioxidant enzymes that have been associated with a variety of oxidative stress-related diseases. Recent data suggest that PON1 is inactivated by oxidative stress, resulting in decreased antioxidant capacity of the individual and aggravation of disease states. Expression of PON2 in brain protects cells from oxidative stress and neuroinflammation generated by neurotoxic compounds. Interestingly, PON2 expression is higher in females than in males, and is upregulated by estrogens. Project 3 consists of four specific aims involved in understanding the roles of PON1 and PON2 in modulating oxidative stress-induced neurotoxicity. Specific Aim 1 will characterize factors that modify the activities of PON1. In Sub-aim 1a kinetic and mass spectrometric analyses will be used to examine modifications of mouse and human recombinant PON1s resulting from in vitro exposures to oxidized lipid metabolites. Sub-aim 1b will examine the effects on PON1 of in vivo exposures of wild type (WT) and PON2-/- mice to Mn and Cd. Specific Aim 2 will examine the role of recombinant mouse PON2 (rMoPON2) in protecting PON1 activity. Sub-aim 2a will explore the fate and function of rMoPON2 incubated with macrophages from PON2-/- mice, which are deficient in modulating oxidative stress. Sub- aim 2b will determine the tissue distribution of rMoPON2 injected into PON2-/- mice. Sub-aim 2c will focus on the efficacy of injected rMoPON2 in preventing the effects of oxidative stress on PON1 in PON2-/- mice exposed to Mn or Cd. Specific Aim 3 will examine the modulation of PON2 levels. Sub-aim 3a will investigate modulation of PON2 levels in the cortical neurons by estrogens with reporter gene assays and the role of microglial PON2 in the neuroprotective effects of estrogens in vitro. Sub-aim 3b will investigate the effect of dopamine on PON2 levels and susceptibility to oxidative stress in cortical neurons. Sub-aim 3c aims to develop a protocol for determining PON2 status by examining the levels of PON2 protein, activity and mRNA in mouse and human macrophages and monocytes. PON2 status in males and in pre- and post-menopausal women will be analyzed. Specific Aim 4 will examine the role of PON2 in modulating susceptibility to the neurotoxicants Mn and Cd. In Sub-aim 4a, neurons and astrocytes from male and female PON2-/- mice will be studied in vitro for resistance to oxidative stress generated by Mn and Cd exposure. In Sub-aim 4b, effects of PON2 genotype and gender on sensitivity to Mn and Cd exposures will be examined in WT and PON2-/- mice with behavioral and biochemical endpoints. Altogether, the proposed experiments will provide novel information on the roles of PON1 and PON2 in modulating oxidative stress and protecting individuals from cardiovascular and neurological diseases.